The peroxisome proliferator-activated receptors (PPARs) form a subfamily in the nuclear receptor superfamily. Three isoforms, encoded by separate genes, have been identified thus far: PPAR[gamma], PPAR[alpha], and PPAR[delta]. The PPARs are ligand-dependent transcription factors that regulate target gene expression by binding to specific peroxisome proliferator response elements (PPREs) in enhancer sites of regulated genes. PPARs possess a modular structure composed of functional domains that include a DNA binding domain (DBD) and a ligand binding domain (LBD). The DBD specifically binds PPREs in the regulatory region of PPAR-responsive genes. The LBD, located in the C-terminal half of the receptor contains the ligand-dependent activation domain, AF-2. Each receptor binds to its PPRE as a heterodimer with a retinoid X receptor (RXR). Upon binding of an agonist, the conformation of a PPAR is altered and stabilized such that a binding cleft, made up in part of the AF-2 domain, is created and recruitment of transcriptional coactivators occurs. Coactivators augment the ability of nuclear receptors to initiate the transcription process. The result of the agonist-induced PPAR-coactivator interaction at the PPRE is an increase in gene transcription. Downregulation of gene expression by PPARs appears to occur through indirect mechanisms (Berger J and Wagner J A, 2002).
PPAR[alpha] is expressed in numerous metabolically active tissues, including liver, kidney, heart, skeletal muscle, and brown fat. It is also present in monocytes, vascular endothelium, and vascular smooth muscle cells. Activation of PPAR[alpha] induces hepatic peroxisome proliferation, hepatomegaly, and hepatocarcinogenesis in rodents. These toxic effects are not observed in humans, although the same compounds activate PPAR[alpha] across species. There are two PPAR[gamma] isoforms expressed at the protein level in mouse and human, [gamma]1 and [gamma]2. They differ only in that the latter has 30 additional amino acids at its N terminus due to differential promoter usage within the same gene, and subsequent alternative RNA processing. PPAR[gamma]2 is expressed primarily in adipose tissue, while PPAR[gamma]1 is expressed in a broad range of tissues. PPAR[delta] is expressed in a wide range of tissues and cells with the highest levels of expression found in the digestive tract, heart, kidney, liver, adipose, and brain.
Kota provides a review of biological mechanisms involving PPARs that includes a discussion of the possibility of using PPAR modulators for treating a variety of conditions, including chronic inflammatory disorders such as atherosclerosis, arthritis and inflammatory bowel syndrome, retinal disorders associated with angiogenesis, increased fertility, and neurodegenerative diseases (Kota B P et al., 2005).
Yousef discusses the anti-inflammatory effects of PPAR[alpha], PPAR[gamma] and PPAR[delta] agonists, suggesting that PPAR agonists may have a role in treating neuronal diseases such as Alzheimer's disease, and autoimmune diseases such as inflammatory bowel disease and multiple sclerosis (Youssef J and Badr M, 2004). A potential role for PPAR agonists in the treatment of Alzheimer's disease has been described in Combs et al., (Combs C K et al., 2000), and such a role for PPAR agonists in Parkinson's disease is discussed in Breidert et al. (Breidert T et al., 2002). A potential related function of PPAR agonists in treatment of Alzheimer's disease, that of regulation of the APP-processing enzyme BACE, has been discussed by Sastre (Sastre M et al., 2003). These studies collectively indicate PPAR agonists may provide advantages in treating a variety of neurodegenerative diseases by acting through complementary mechanisms.
Discussion of the anti-inflammatory effects of PPAR agonists is also available in Feinstein et al., (Feinstein D L, 2004), in relation to multiple sclerosis and Alzheimer's disease; Patel et al., (Patel H J et al., 2003) in relation to chronic obstructive pulmonary disease (COPD) and asthma; Lovett-Racke et al., (Lovett-Racke A E et al., 2004) in relation to autoimmune disease; Malhotra et al., (Malhotra S et al., 2005) in relation to psoriasis; and Storer et al., (Storer P D et al., 2005) in relation to multiple sclerosis.
This wide range of roles for the PPARs that have been discovered suggest that PPAR[alpha], PPAR[gamma] and PPAR[delta] play a role in a wide range of events involving the vasculature, including atherosclerotic plaque formation and stability, thrombosis, vascular tone, angiogenesis, cancer, pregnancy, pulmonary disease, autoimmune disease, and neurological disorders.
The fibrates, amphipathic carboxylic acids that have been proven useful in the treatment of hypertriglyceridemia, are PPAR[alpha] ligands. Clofibrate and fenofibrate have been shown to activate PPARa with a 10-fold selectivity over PPAR[gamma]. Bezafibrate acts as a pan-agonist that shows similar potency on all three PPAR isoforms. Fibrates are known to regulate expression of genes (acyl CoA synthase, lipoprotein lipase, fatty acid transport protein and the like) relating to the metabolism of fatty acid and apolipoprotein (AI, AII, AV, CIII) genes involved in triglyceride (TG) and cholesterol metabolism, by activation of PPAR[alpha], decreases TG and LDL cholesterol and increases HDL cholesterol (Bocher V et al., 2002, Lefebvre P et al., 2006). Thus, fenofibrate is known to be highly effective as a therapeutic drug for hyperlipidemia. PPAR[alpha] also exerts anti-inflammatory and antiproliferative effects and prevents the proatherogenic effects of accumulation of cholesterol in macrophages by stimulating the outflow of cholesterol (Lefebvre P et al). Fenofibrate significantly reduced proteinuria, inflammatory cell recruitment and extracellular matrix (ECM) proteins deposition in the kidney of hypertensive SHR rats without apparent effect on blood pressure. A marked reduction of oxidative stress accompanied by reduced activity of renal NAD(P)H oxidase, increased activity of Cu/Zn SOD, and decreased phosphorylation of p38MAPK and JNK was detected in the kidney of fenofibrate treated SHR rat (Hou X et al., 2010). Fenofibrate significantly reduced superoxide production, protein oxidation and infarct size in the ischemic brain at 30 minutes after reperfusion (Wang G et al., 2010). Fenofibrate administration significantly decreased the cerebral infarct volume and reduced microglial activation and neutrophil infiltration into the ischaemic zone (Ouk T et al., 2009). This effect was associated with partial prevention of post-ischaemic endothelial dysfunction.
The finding that the thiazolidinediones mediate their therapeutic effects through direct interactions with PPAR[gamma] established this target as a key regulator of glucose and lipid homeostasis. PPAR[gamma] improves insulin resistance and thereby has a hypoglycemic effect. Ligands known for PPAR[gamma] include synthetic compounds such as unsaturated fatty acids (e.g., [alpha]-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid) and thiazolidine-type antidiabetic drugs (e.g., troglitazone, pioglitazone, rosiglitazone) (Bhatia V and Viswanathan P, 2006, Nagy L et al., 1998). These ligands are known to suppress hyperplasia of large adipocytes and to increase the number of insulin-sensitive small adipocytes, so that they improve insulin resistance and thereby reduce blood glucose levels (Tontonoz P and Spiegelman B M, 2008, Walczak R and Tontonoz P, 2002).
One of the earliest findings associating PPARs and macrophages was that PPAR[gamma] was highly expressed in macrophage-derived foam cells of human and murine atherosclerotic lesions. Subsequently, it has been demonstrated that PPAR[gamma] is expressed in human and murine monocytes/macrophages. Functionally, PPAR[gamma] has been shown to play a role in the differentiation and activation of monocytes and in the regulation of inflammatory activities (Chawla A et al., 2001, Li A C et al., 2004). Many studies have demonstrated that PPAR[gamma] ligands inhibit macrophage-mediated inflammatory responses. Thiazolidinediones have been found to inhibit the secretion of many of these mediators (including gelatinase B, IL-6, TNF-a, and IL-1) and also to reduce the induced expression of inducible NOS (iNOS) and the transcription of the scavenger receptor (Chawla A et al., 2001, Li A C et al., 2004).
The relevance of PPAR[gamma] has been studied in several human autoimmune diseases and animal models of autoimmune diseases. Kawahito et al. demonstrated that synovial tissue expressed PPAR[gamma] in patients with rheumatoid arthritis (Kawahito Y et al., 2000). PPAR[gamma] was found to be highly expressed in macrophages, and modest expression was noted in synovial-lining fibroblasts and ECs. Activation of PPAR[gamma] by 15d-PGJ2 and troglitazone induced RA synoviocyte apoptosis in vitro. It has been suggested that PPAR[gamma] is functionally relevant in freshly isolated T cells or becomes functionally relevant early in activation. In these studies, it was also demonstrated that the two ligands for PPAR[gamma] mediated inhibition of IL-2 secretion by the T-cell clones and did not inhibit IL-2-induced proliferation of such clones. Several studies have investigated the role of PPAR[gamma] ligands in modifying animal models of autoimmune diseases. Su et al. showed that in a mouse model of inflammatory bowel disease, thiazolidinediones markedly reduced colonic inflammation (Su C G et al., 1999). It has been proposed that this effect might be a result of a direct effect on colonic epithelial cells, which express high levels of PPAR[gamma] and can produce inflammatory cytokines. Kawahito et al. demonstrated that intraperitoneal administration of the PPAR[gamma] ligands, 15d-PGJ2 and troglitazone, ameliorated adjuvant-induced arthritis (Kawahito Y et al., 2000). Niino and Feinstein examined the effect of a thiazolidinedione on experimental allergic encephalomyelitis and found that this treatment attenuated the inflammation and decreased the clinical symptoms in this mouse model of multiple sclerosis (Feinstein D L et al., 2002, Niino M et al., 2001).
Alzheimer's disease (AD) is characterized by the extracellular deposition of beta-amyloid fibrils within the brain and the activation of microglial cells associated with the amyloid plaque. The activated microglia subsequently secrete a diverse range of inflammatory products. Kitamura et al. assessed the occurrence of PPAR[gamma] and COX-1, COX-2, in normal and AD brains using specific antibodies and found increased expression of these moieties in AD brains (Kitamura Y et al., 1999). Nonsteroidal, anti-inflammatory drugs (NSAIDs) have been shown to be efficacious in reducing the incidence and risk of AD and in delaying disease progression. Combs et al. demonstrated that NSAIDs, thiazolidinediones, and PGJ2, all of which are PPAR[gamma] agonists, inhibited the beta-amyloid-stimulated secretion of inflammatory products by microglia and monocytes. PPAR[gamma] agonists were shown to inhibit the beta-amyloid-stimulated expression of the genes for IL-6 and TNFa and the expression of COX-2 (Combs C K et al., 2000). Heneka et al. demonstrated that microinjection of LPS and IFN-a into rat cerebellum induced iNOS expression in cerebellar granule cells and subsequent cell death (Heneka M T et al., 2000). Coinjection of PPAR[gamma] agonists (including troglitazone and 15d-PGJ2) reduced iNOS expression and cell death, whereas coinjection of a selective COX inhibitor had no effect. Overall, work in AD seems to suggest that PPAR[gamma] agonists can modulate inflammatory responses in the brain and that NSAIDs may be helpful in AD as a result of their effect on PPAR[gamma].
The low dose combination of fenofibrate and rosiglitazone was more effective in attenuating the diabetes-induced experimental nephropathy and renal oxidative stress as compared to treatment with either drug alone or lisinopril (Arora M K et al., 2010). The concurrent administration of fenofibrate and rosiglitazone at low doses may have prevented the development of diabetes induced nephropathy by reducing the lipid alteration, decreasing the renal oxidative stress and certainly providing the direct nephroprotective action.
PPAR ligands have also been identified as dual PPAR[gamma]/[alpha] agonists. By virtue of the additional PPAR[alpha] agonist activity, this class of compounds has potent lipid-altering efficacy in addition to antihyperglycemic activity in animal models of lipid disorders. KRP-297 is an example of a TZD dual PPAR[gamma]/[alpha] agonist (Murakami K et al., 1998); furthermore DRF-2725 and AZ-242 are non-TZD dual PPAR[gamma]/[alpha] agonists (Cronet P et al., 2001, Lohray B B et al., 2001).
Recently, potent PPAR[delta] ligands have been published allowing a better understanding of its function in lipid metabolism (Barak Y et al., 2002, Oliver W R, Jr. et al., 2001, Tanaka T et al., 2003, Wang Y X et al., 2003). The main effect of these compounds in db/db mice (Leibowitz M D et al., 2000) and obese rhesus monkeys (Oliver W R, Jr. et al., 2001) was an increase of high density lipoprotein cholesterol (HDL-C) and a decrease in triglycerides with little effect on glucose (although insulin levels were decreased in monkeys). HDL-C serves to remove cholesterol from peripheral cells through a process called reverse cholesterol transport. The first and rate-limiting step, which is a transfer of cellular cholesterol and phospholipids to the apolipoprotein A-I component of HDL3 is mediated by the ATP binding cassette transporter A1 (ABCA1) (Lawn R M et al., 1999). PPAR[delta] activation appears to increase HDL-C through transcriptional regulation of ABCA1 (Oliver W R, Jr. et al., 2001). Therefore, by inducing ABCA1 mRNA in macrophages, PPAR[delta] agonists could increase HDL-C levels in patients and remove excess cholesterol from lipid-laden macrophages, one of the major players in atherosclerotic lesion development. This would be an alternative therapy to the statin drugs, which show little effect on HDL-C and mainly decrease LDL-C or the fibrates, the only marketed PPAR[alpha] agonists, having low potency and inducing only modest HDL-C elevations. In addition, like the fibrates, PPAR[delta] agonists have the potential to also reduce triglycerides, an additional risk factor for cardiovascular disease.
PPAR[delta] is highly expressed in skeletal muscle cells, and further PPAR[delta] is involved in the expression of genes associated with fatty acid metabolism and has the function of stimulating fatty acid metabolism in skeletal muscle cells or fat tissue. PPAR[delta] conditional knock-out mice, engineered to lack receptor expression specifically in the myogenic cells, had 40% fewer satellite cells than their wild-type littermates, and these satellite cells exhibited reduced growth kinetics and proliferation in vitro (Angione A R et al., 2011). Furthermore, regeneration of PPAR[delta] muscles was impaired after cardiotoxin-induced injury. These results support a function of PPAR[delta] in regulating skeletal muscle metabolism and insulin sensitivity. In-line with these findings, transgenic mice designed to overexpress PPAR[delta] in their skeletal muscle are less likely to develop high-fat diet-induced obesity or insulin resistance, and their adipocytes become smaller in size.
By various other mechanisms, PPAR[delta] agonists are effective at preventing, reversing, or treating other types of inflammations and particularly diseases linked to lung inflammation. Using intravital microscopy in the mouse cremasteric microcirculation, Piqueras et al., have shown that activation of PPAR[delta] by its selective ligand GW501516 inhibited TNF-alpha induced leukocyte rolling flux, adhesion, and emigration in a dose-dependent manner (Piqueras L et al., 2009). Moreover, PPAR[delta] agonists reduced the expression of adhesion molecules such as ICAM-1, VCAM-1, and E-selectin in the cremasteric postcapillary venules. Similarly, rolling and adhesion of hPMNs under physiological flow on TNF-alpha-activated HUVECs were also inhibited markedly by GW501516. These inhibitory responses of GW501516 on activated endothelium were accompanied by a reduction in TNF-alpha induced endothelial GRO-release and VCAM-1, E-selectin, and ICAM-1 mRNA expression. Taken together, these results show that PPAR [delta] modulates acute inflammation in vivo and in vitro under flow by targeting the neutrophil-endothelial cell (Piqueras L et al., 2009).
Renal ischemia, also called nephric ischemia, is the deficiency of blood in one or both kidneys, or nephrons, usually due to functional constriction or actual obstruction of a blood vessel. Acute renal ischemia is associated with significant morbidity and mortality. There has been little progress in treating the disease over the last 50 years. Currently dialysis is the only effective therapy. A few reports have proposed a relationship between the activation of PPAR[alpha] (Portilla D et al., 2000), PPAR[gamma] (Sivarajah A et al., 2003) and PPAR[delta] (Letavernier E et al., 2005) and protection from acute renal ischemia. It has been suggested that the protective effect of PPAR[delta] may be due to its activation of the anti-apoptotic Akt signaling pathway and by promoting increased spreading of tubular epithelial cells.
Examples of known PPAR delta agonists variously useful for hyperlipidemia, diabetes, or atherosclerosis include L-165041 (Leibowitz M D et al., 2000) and GW501516 (Oliver W R, Jr. et al., 2001). There is a further need for new PPAR delta agonists for the treatment of diabetes, nephropathy, neuropathy, retinopathy, polycystic ovary syndrome, hypertension, ischemia, stroke, irritable bowel disorder, inflammation, cataract, cardiovascular diseases, metabolic syndrome, X syndrome, hyper-LDL-cholesterolemia, dyslipidemia (including hypertriglyceridemia, hypercholesterolemia, mixed hyperlipidemia, and hypo-HDL-cholesterolemia), atherosclerosis, obesity, and other disorders related to lipid metabolism and energy homeostasis complications thereof.
The old and well known lipid-lowering fibric acid derivative bezafibrate is the first clinically tested panPPAR activator. Bezafibrate leads to considerable raising of HDL cholesterol and reduces triglycerides, improves insulin sensitivity and reduces blood glucose level, significantly lowering the incidence of cardiovascular events and new diabetes in patients with features of metabolic syndrome (Tenenbaum A et al., 2005). Clinical evidences obtained from bezafibrate-based studies strongly support the concept of pan-PPAR therapeutic approach to conditions which comprise the metabolic syndrome.
Both bezafibrate and GW501516 inhibited the methionine- and choline-deficient (MCD)-diet-induced elevations of hepatic triglyceride and thiobarbituric acid-reactants contents and the histopathological increases in fatty droplets within hepatocytes, liver inflammation and number of activated hepatic stellate cells (Nagasawa T et al., 2006). In this model, both ligands increased the levels of hepatic mRNAs associated with fatty acid beta-oxidation and reduced the levels of those associated with inflammatory cytokines or chemokine. In addition, bezafibrate characteristically reduced the elevation in the level of plasma ALT, but enhanced that in plasma adiponectin and increased the mRNA expression levels of its receptors. These results suggest that panPPAR activators may improve non-alcoholic steatohepatitis.
The results of the Bezafibrate Infarction Prevention (BIP) Study demonstrated that in diabetic patients, bezafibrate administration over two years period prevented a progressive decline of beta cell function and an increase of insulin resistance (Tenenbaum H et al., 2007). Bezafibrate therapy in the BIP trial was also associated with significant long-term cardiovascular protection despite the unbalanced usage of nonstudy lipid lowering drugs during the course of the trial (Goldenberg I et al., 2008). The results of the 16-year mortality follow-up of the BIP trial demonstrated that patients allocated to bezafibrate therapy experienced a significant 11% reduction in the risk of long-term mortality compared with placebo-allocated patients (Goldenberg I et al., 2009).